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/content/aip/journal/bmf/3/1/10.1063/1.3098319
2009-03-16
2016-12-09

Abstract

Two microfluidic systems have been developed for specific analysis of -glutamate in food based on substrate recycling fluorescence detection. -glutamate dehydrogenase and a novel enzyme,-phenylglycine aminotransferase, were covalently immobilized on (i) the surface of silicon microchips containing 32 porous flow channels of depth and width and (ii) polystyrene Poros™ beads with a particle size of . The immobilized enzymes recycle -glutamate by oxidation to 2-oxoglutarate followed by the transfer of an amino group from -4-hydroxyphenylglycine to 2-oxoglutarate. The reaction was accompanied by reduction of nicotinamide adenine dinucleotide () to NADH, which was monitored by fluorescence detection . First, the microchip-based system, -glutamate was detected within a range of 3.1–50.0 mM. Second, to be automatically determined, sequential injection analysis (SIA) with the bead-based system was investigated. The bead-based system was evaluated by both flow injection analysis and SIA modes, where good reproducibility for -glutamate calibrations was obtained (relative standard deviation of 3.3% and 6.6%, respectively). In the case of SIA, the beads were introduced and removed from the microchip automatically. The immobilized beads could be stored in a 20% glycerol and 0.5 mM ethylenediaminetetraacetic acid solution maintained at a of 7.0 using a phosphate buffer for at least 15 days with 72% of the activity remaining. The bead-based system demonstrated high selectivity, where -glutamate recoveries were between 91% and 108% in the presence of six other -amino acids tested.

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